The long term goals of this proposal are to construct spatially defined arrays of oligonucleotide probes and to study the feasibility of using these arrays in applications of sequencing DNA by hybridization. A multidisciplinary research program is proposed which will integrate the necessary expertise in photolithography, photochemistry, synthetic chemistry, detection technology, informatics and applications to large scale DNA sequencing. We will apply newly developed techniques in light-directed polymer synthesis to oligonucleotide chemistry, explore kinetic and solvent related parameters of target hybridization to oligonucleotide arrays, read the positions of hybridization by epifluorescence microscopy, and apply new combinatorial methods to determine sequence from the hybridization data. The method will be applied to actual sequencing applications at the yeast genome center. Successful completion of this work will lead to sequencing instrumentation that will provide order of magnitude improvements in DNA sequencing productivity and will be directly applicable to the Human Genome Project.